CRISPR/cas genome editing for neurodegenerative diseases: Mechanisms, therapeutic advances, and clinical prospects.

Anzalone Prime: An Interview with Prime Editing Developer Andrew Anzalone

Neurodegenerative diseases (NDDs) are currently the most widespread neuronal pathologies in the world. Among these, the most widespread are Alzheimer's disease (AD), dementia, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD)-all characterized by a progressive loss of neurons in specific regions of the brain leading to varied clinical symptoms. At the basis of neurodegenerative diseases, an emerging role is played by genetic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene that cause increased LRRK2 activity with consequent alteration of neuronal autophagy pathways. LRRK2 kinase activity requires GTPase activity which functions independently of kinase activity and is required for neurotoxicity and to potentiate neuronal death. Important in the neurodegeneration process is the upregulation of casein kinase (CK), which causes the alteration of the AMPK pathway by enhancing the phosphorylation of α-synuclein and huntingtin proteins, known to be involved in PD and HD, and increasing the accumulation of the amyloid-β protein (Aβ) for AD. Recent research has identified CK of the kinases upstream of LRRK2 as a regulator of the stability of the LRRK2 protein. Based on this evidence, this review aims to understand the direct involvement of individual kinases in NDDs and how their crosstalk may impact the pathogenesis and early onset of neurodegenerative diseases.

Activation of Nrf2 signaling as a common treatment of neurodegenerative diseases.

Neurotransmitter transporters (NTTs) control synaptic responses by modulating the concentration of neurotransmitters at the synaptic cleft. Glutamate is the most abundant excitatory neurotransmitter in the brain and needs to be finely tuned in time and space to maintain a healthy brain and precise neurotransmission. The glutamate transporter EAAT2 (SLC1A2) is primarily responsible for glutamate clearance. EAAT2 impairment has been associated with Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to both monogenic and sporadic forms of PD, of which the common substitution Gly2019Ser is associated with a significant deficit in EAAT2 expression. The role of pathological mutants of the LRRK2 is intensively studied and reviewed. Here we have focused the attention on the physiological role of LRRK2 on EAAT2, comparing the activity of NTTs with or without the LRRK2 kinase. By heterologous expression in Xenopus laevis oocytes and two-electrode voltage clamp, the current amplitudes of the selected NTTs and kinetic parameters have been collected in the presence and absence of LRRK2. The results show that EAAT2 expression and function are impaired in the absence of the kinase and also under its pharmacological inhibition via MLi-2 treatment. LRRK2 stabilizes EAAT2 expression increasing the amount of transporter at the plasma membrane. Interestingly, the LRRK2 action is EAAT2-specific, as we observed no significant changes in the transport current amplitude and kinetic parameters obtained for the other excitatory and inhibitory NTTs studied. This study, for the first time, demonstrates the physiological importance of LRRK2 in EAAT2 function, highlighting the specificity of LRRK2-mediated modulation of EAAT2 and suggesting a potential role for the kinase as a checkpoint for preserving neurons from excitotoxicity. In brain conditions associated with impaired glutamate clearance, targeting LRRK2 for EAAT2 regulation may offer novel therapeutic opportunities.

Dominant missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson disease. LRRK2 encodes a serine/threonine protein kinase, and pathogenic mutations may increase kinase activity. Intrinsic GTP binding in the GTPase domain may govern kinase activity through an internal signal transduction cascade. As with many protein kinases, LRRK2 self-interacts through mechanisms that may regulate enzymatic activity. We find that the disruption of either GTPase or kinase activity enhances the formation of high molecular weight oligomers and prevents the formation of LRRK2 dimer structures. In addition, brief application of the broad spectrum kinase inhibitor staurosporine ablates LRRK2 dimers and promotes LRRK2 high molecular weight oligomers. LRRK2 interactions with other proteins in cell lines are kinase-independent and include chaperones and cell cytoskeleton components, suggesting that LRRK2 self-assembly principally dictates complex size. To further explore the mechanics of kinase activation, we separate soluble LRRK2 protein that encodes the pathogenic G2019S mutation into high molecular weight oligomers, dimers, and monomers and find that kinase activity resides with dimeric LRRK2. Some PD-associated mutations that increase kinase activity in vitro significantly increase the proportion of dimer structures relative to total LRRK2 protein, providing additional insight into how pathogenic mutations may alter normal enzymatic regulation. Targeting and tracking LRRK2 dimerization may provide a clear way to observe LRRK2 kinase activity in living cells, and disruption of dimeric LRRK2 through kinase inhibition or other means may attenuate pathogenic increases in LRRK2 enzymatic output.

Therapeutic Translation of iPSCs for Treating Neurological Disease

Recent advancements in stem cell and gene therapies for neurological disorders and intractable epilepsy

To diagnose neurodegenerative diseases (NDDs), physicians have been clinically evaluating symptoms. However, these symptoms are not very dependable—particularly in the early stages of the diseases. This study has therefore proposed a novel classification algorithm that uses a deep learning approach to classify NDDs based on the recurrence plot of gait vertical ground reaction force (vGRF) data. The irregular gait patterns of NDDs exhibited by vGRF data can indicate different variations of force patterns compared with healthy controls (HC). The classification algorithm in this study comprises three processes: a preprocessing, feature transformation and classification. In the preprocessing process, the 5-min vGRF data divided into 10-s successive time windows. In the feature transformation process, the time-domain vGRF data are modified into an image using a recurrence plot. The total recurrence plots are 1312 plots for HC (16 subjects), 1066 plots for ALS (13 patients), 1230 plots for PD (15 patients) and 1640 plots for HD (20 subjects). The principal component analysis (PCA) is used in this stage for feature enhancement. Lastly, the convolutional neural network (CNN), as a deep learning classifier, is employed in the classification process and evaluated using the leave-one-out cross-validation (LOOCV). Gait data from HC subjects and patients with amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD) and Parkinson’s disease (PD) obtained from the PhysioNet Gait Dynamics in Neurodegenerative disease were used to validate the proposed algorithm. The experimental results included two-class and multiclass classifications. In the two-class classification, the results included classification of the NDD and the HC groups and classification among the NDDs. The classification accuracy for (HC vs. ALS), (HC vs. HD), (HC vs. PD), (ALS vs. PD), (ALS vs. HD), (PD vs. HD) and (NDDs vs. HC) were 100%, 98.41%, 100%, 95.95%, 100%, 97.25% and 98.91%, respectively. In the multiclass classification, a four-class gait classification among HC, ALS, PD and HD was conducted and the classification accuracy of HC, ALS, PD and HD were 98.99%, 98.32%, 97.41% and 96.74%, respectively. The proposed method can achieve high accuracy compare to the existing results, but with shorter length of input signal (Input of existing literature using the same database is 5-min gait signal, but the proposed method only needs 10-s gait signal).

Editorial: A Trends guide to Neurodegenerative Disease and Repair

Human neurological diseases such as Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple sclerosis (MS), stroke and spinal cord injury (SCI) are caused by loss of neurons and glia in the brain or spinal cord. Cell replacement therapy and gene transfer to the diseased or injured brain have provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases. However, the paucity of suitable cell types for cell replacement therapy in patients suffering from neurological disorders has hampered the development of this promising therapeutic approach. In recent years, neurons and glia have successfully been generated from stem cells such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs), and extensive efforts by investigators to develop stem cell-based brain transplantation therapies have been carried out. I review here notable experimental and pre-clinical studies previously published involving stem cell-based cell- and gene-therapies for PD, HD, ALS, AD, MS, stroke and SCI, and discuss for future prospect for the stem cell therapy of neurological disorders in clinical setting. There are still many obstacles to be overcome before clinical application of cell- and gene-therapy in neurological disease patients is adopted: (i) it is still uncertain how to generate specific cell types of neurons or glia suitable for cellular grafts in great quantity, (ii) it is required to abate safety concern related to tumor formation following NSC transplantation, and (iii) it needs to be better understood by what mechanism transplantation of NSCs leads to an enhanced functional recovery. Steady and stepwise progress in stem cell research in both basic and pre-clinical settings should support the hope for development of stem cell-based therapies for neurodegenerative diseases. This review focuses on the utility of stem cells particularly NSCs as substrates for structural and functional repair of the diseased or injured brain.

Background While Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS) are clinically distinct neurodegenerative disorders, there have been reports of the co-occurrence of the two conditions in single individuals. Nonetheless, to our knowledge there has been no systematic review of the co-occurrence of HD and ALS to summarize the evidence to date. Aims 1) To systematically review the literature on the co-occurrence of HD and ALS in single individuals. 2) To examine the common features among the cases of individuals with HD and ALS. 3) To determine if the two disorders occurred together coincidentally or if they are causally related through an underlying mechanism. Methods A systematic review of all the literature on the co-occurrence of HD and ALS in single individuals – including case reports, observational studies, clinical trials, as well as mechanistic studies – until May 31, 2018 was performed, and descriptive analyses were conducted. Results Our searches have revealed 15 cases of individuals with confirmed diagnoses of both HD and ALS reported in the literature to date – and at least 5 other probable cases of HD and ALS. Among these 15 confirmed cases, the majority (13/15 cases) had an age of onset of HD symptoms between 40 and 72 years – generally higher than the average age of onset for individuals with HD without ALS. These cases had 39–47 CAG repeats in the Huntington gene – the lower end of the numbers of repeats in HD patients. More than half of these cases (9/15) had a diagnosis for ALS ranging from several months to years after the diagnosis of HD; 2/15 cases had a diagnosis of ALS before the diagnosis of HD (by 7 to 12 months); and 2/15 cases had ALS and HD diagnosed, or had symptoms of both, around the same time. There have been no observational studies or clinical trials on the co-occurrence of HD and ALS to date. Two mechanistic studies on the co-occurrence of HD and ALS were identified. Conclusion The number of reported cases on the co-occurrence of HD and ALS remains very low, which has rendered it highly challenging to conduct observational studies or clinical trials. Nonetheless, the current systematic review has revealed some common features among the reported cases, and has reviewed the proposed mechanisms of such co-occurrence. Further studies are warranted, and may be enhanced by the involvements of international consortia on HD and ALS.

Membrane lipid peroxidation and oxidative modification of various membrane and associated proteins (e.g., receptors, ion transporters and channels, and signal transduction and cytoskeletal proteins) occur in a range of neurodegenerative disorders. This membrane-associated oxidative stress (MAOS) is promoted by redox-active metals, most notably iron and copper. The mechanisms whereby different genetic and environmental factors initiate MAOS in specific neurological disorders are being elucidated. In Alzheimer's disease (AD), the amyloid beta-peptide generates reactive oxygen species and induces MAOS, resulting in disruption of cellular calcium homeostasis. In Parkinson's disease (PD), mitochondrial toxins and perturbed ubiquitin-dependent proteolysis may impair ATP production and increase oxyradical production and MAOS. The inheritance of polyglutamine-expanded huntingtin may promote neuronal degeneration in Huntington's disease (HD), in part, by increasing MAOS. Increased MAOS occurs in amyotrophic lateral sclerosis (ALS) as the result of genetic abnormalities (e.g., Cu/Zn-superoxide dismutase mutations) or exposure to environmental toxins. Levels of iron are increased in vulnerable neuronal populations in AD and PD, and dietary and pharmacological manipulations of iron and copper modify the course of the disease in mouse models of AD and PD in ways that suggest a role for these metals in disease pathogenesis. An increasing number of pharmacological and dietary interventions are being identified that can suppress MAOS and neuronal damage and improve functional outcome in animal models of AD, PD, HD, and ALS. Novel preventative and therapeutic approaches for neurodegenerative disorders are emerging from basic research on the molecular and cellular actions of metals and MAOS in neural cells.

BackgroundThe semiology and determinants of apathy are largely unknown across amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Huntington’s disease (HD), due to both motor and non-motor confounders. This study thus aimed at (1) profiling apathy in ALS, PD, and HD and (2) exploring its clinical determinants.MaterialsConsecutive ALS (N = 99), PD (N = 73), and HD (N = 25) patients underwent a motor-free assessment of apathy (Dimensional Apathy Scale, DAS), global cognition, anxiety and depression. Function was assessed through disease-specific scales. The DAS was also completed by N = 101 healthy controls (HCs). Between-group comparisons on DAS scores were implemented by covarying for all applicable confounders. Predictive models on DAS scores were built through multiple, stepwise regressions.ResultsParkinson’s disease and HD, but not ALS, patients were more apathetic than HCs—with HD patients also selectively showing lower initiation and poorer goal-directed planning than HCs. Higher apathetic features were detected in PD and HD as compared to ALS. Education was a protective factor against apathy in ALS. Anxiety was a risk factor for global apathy in ALS, HD, and to a lesser extent, in PD, whereas, protective only toward affective disintegration in PD and ALS. Cognitive inefficiency was a risk factor toward apathy in both PD and ALS. Depression was a risk factor for executive-related apathy in PD.DiscussionThis study provides unprecedented insights into the heterogeneous semiology and determinants of apathy across ALS, PD, and HD via the DAS, in turn informing clinical practice and research.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial and apparently sporadic Parkinson disease. LRRK2 is a multidomain protein kinase with autophosphorylation activity. It has previously been shown that the kinase activity of LRRK2 is required for neuronal toxicity, suggesting that understanding the mechanism of kinase activation and regulation may be important for the development of specific kinase inhibitors for Parkinson disease treatment. Here, we show that LRRK2 predominantly exists as a dimer under native conditions, a state that appears to be stabilized by multiple domain-domain interactions. Furthermore, an intact C terminus, but not N terminus, is required for autophosphorylation activity. We identify two residues in the activation loop that contribute to the regulation of LRRK2 autophosphorylation. Finally, we demonstrate that LRRK2 undergoes intramolecular autophosphorylation. Together, these results provide insight into the mechanism and regulation of LRRK2 kinase activity.

The impact of feature extraction for the classification of amyotrophic lateral sclerosis among neurodegenerative diseases and healthy subjects